Herpes virus entry mediator signaling blockade produces mortality in neonatal sepsis through induced cardiac dysfunction.

Introduction: Sepsis remains a major source of morbidity and mortality in neonates, and characterization of immune regulation in the neonatal septic response remains limited. HVEM is a checkpoint regulator which can both stimulate or inhibit immune responses and demonstrates altered expression after sepsis. We hypothesized that signaling via HVEM would be essential for the neonatal response to sepsis, and that therefore blockade of this pathway would improve survival to septic challenge. Methods: To explore this, neonatal mice were treated with cecal slurry (CS), CS with Anti-HVEM antibody (CS-Ab) or CS with isotype (CS-IT) and followed for 7-day survival. Mice from all treatment groups had thymus, lung, kidney and peritoneal fluid harvested, weighed, and stained for histologic evaluation, and changes in cardiac function were assessed with echocardiography. Results: Mortality was significantly higher for CS-Ab mice (72.2%) than for CS-IT mice (22.2%). CS resulted in dysregulated alveolar remodeling, but CS-Ab lungs demonstrated significantly less dysfunctional alveolar remodeling than CS alone (MCL 121.0 CS vs. 87.6 CS-Ab), as well as increased renal tubular vacuolization. No morphologic differences in alveolar septation or thymic karyorrhexis were found between CS-Ab and CS-IT. CS-Ab pups exhibited a marked decrease in heart rate (390.3 Sh vs. 342.1 CS-Ab), stroke volume (13.08 CS-IT vs. 8.83 CS-Ab) and ultimately cardiac output (4.90 Sh vs. 3.02 CS-Ab) as well as a significant increase in ejection fraction (73.74 Sh vs. 83.75 CS-Ab) and cardiac strain (40.74 Sh vs. 51.16 CS-Ab) as compared to CS-IT or Sham animals. Discussion: While receptor ligation of aspects of HVEM signaling, via antibody blockade, appears to mitigate aspects of lung injury and thymic involution, stimulatory signaling via HVEM still seems to be necessary for vascular and hemodynamic resilience and overall neonatal mouse survival in response to this experimental polymicrobial septic insult. This dissonance in the activity of anti-HVEM neutralizing antibody in neonatal animals speaks to the differences in how septic cardiac dysfunction should be considered and approached in the neonatal population.

Severity of critical illness correlates with CD3-low expression on iNKT-cells among septic surgical patients.

BACKGROUND: iNKT-cells are innate regulatory lymphocytes capable of directing immune and inflammatory responses to sepsis. Repeat stimulation of iNKT-cells leads to the induction of anergy with the emergence of a hyporesponsive CD3low iNKT-cell subpopulation. METHODS: iNKT-cells were isolated from critical ill surgical patients with sepsis and phenotyped for CD3 expression. This was correlated with degree of severity of illness, as denoted by APACHE-II score. RESULTS: Comparing healthy volunteers to critically ill septic patients, it was noted that increasing severity of sepsis was associated with increasing frequency of circulating CD3low-iNKT-cell populations. CONCLUSION: The emergence of CD3low -iNKT-cells may serve as a clinically translatable marker of degree of sepsis-induced immune dysfunction.

VISTA Non-redundantly Regulates Proliferation and CD69low γδ T cell Accumulation in the Intestine in Murine Sepsis.

Sepsis is a dysregulated systemic immune response to infection that is responsible for ∼35% of in-hospital deaths at a significant fiscal health care cost. Our laboratory, among others, has demonstrated the efficacy of targeting negative checkpoint regulators (NCRs) to improve survival in a murine model of sepsis, cecal ligation and puncture (CLP). B7-CD28 superfamily member, V-domain Immunoglobulin Suppressor of T cell Activation (VISTA), is an ideal candidate for strategic targeting in sepsis. VISTA is a 35-45 kDa type 1 transmembrane protein with unique biology that sets it apart from all other NCRs. We recently reported that VISTA-/- mice had a significant survival deficit post CLP which was rescued upon adoptive transfer of a VISTA-expressing pMSCV-mouse Foxp3-EF1α-GFP-T2A-puro stable Jurkat cell line (Jurkatfoxp3 T cells). Based on our prior study, we investigated the effector cell target of Jurkatfoxp3 T cells in VISTA-/- mice. γδ T cells are a powerful lymphoid subpopulation that require regulatory fine-tuning by Tregs to prevent overt inflammation/pathology. In this study, we hypothesized that Jurkatfoxp3 T cells non-redundantly modulate the γδ T cell population post CLP. We found that VISTA-/- mice have an increased accumulation of intestinal CD69low γδ T cells which are not protective in murine sepsis. Adoptive transfer of Jurkatfoxp3 T cells, decreased the intestinal γδ T cell population, suppressed proliferation, skewed remaining γδ T cells toward a CD69high phenotype, and increased sCD40L in VISTA-/- mice post CLP. These results support a potential regulatory mechanism by which VISTA skews intestinal γδ T cell lineage representation in murine sepsis.

SPLENIC INVARIANT NATURAL KILLER T CELLS PLAY A SIGNIFICANT ROLE IN THE RESPONSE TO POLYMICROBIAL SEPSIS.

ABSTRACT: Background: Sepsis is marked by a dysregulated immune response to an infection. Invariant natural killer T cells ( i NKT cells) are a pluripotent lymphocyte subpopulation capable of affecting and coordinating the immune response to sepsis. The spleen is an important site of immune interactions in response to an infection. Splenic i NKT cells have emerged as important potential frontline mediators of chronic immune response. There are few data addressing the role splenic of i NKT cells in response to intra-abdominal polymicrobial sepsis. Methods: The cecal ligation and puncture model was used to create intra-abdominal sepsis in 8- to 12-week-old wild-type, i NKT -/- , or programmed cell death receptor-1 (PD-1) -/- mice. Twenty-four hours later, spleens were harvested. Flow cytometry was used for phenotyping using monoclonal antibodies. Cell sort was used to isolate i NKT cells. A macrophage cell line was used to assess i NKT cell-phagocyte interactions. Enzyme-linked immunosorbent assay was used for cytokine analysis. Results: Splenic i NKT-cell populations rapidly declined following induction of sepsis. Within i NKT-cell -/- mice, a distinct baseline hyperinflammatory environment was noted. Within wild type, sepsis induced an increase in splenic IL-6 and IL-10, whereas in i NKT -/- mice, there was no change in elevated IL-6 levels and a noted decrease in IL-10 expression. Further, following sepsis, PD-1 expression was increased upon spleen i NKT cells. With respect to PD-1 ligands upon phagocytes, PD-1 ligand expression was unaffected, whereas PD-L2 expression was significantly affected by the presence of PD-1. Conclusions: Invariant natural killer T cells play a distinct role in the spleen response to sepsis, an effect mediated by the checkpoint protein PD-1. Given that modulators are available in clinical trials, this offers a potential therapeutic target in the setting of sepsis-induced immune dysfunction.

Lymphocyte HVEM/BTLA co-expression after critical illness demonstrates severity indiscriminate upregulation, impacting critical illness-induced immunosuppression.

Introduction: The co-regulatory molecule, HVEM, can stimulate or inhibit immune function, but when co-expressed with BTLA, forms an inert complex preventing signaling. Altered HVEM or BTLA expression, separately have been associated with increased nosocomial infections in critical illness. Given that severe injury induces immunosuppression, we hypothesized that varying severity of shock and sepsis in murine models and critically ill patients would induce variable increases in HVEM/BTLA leukocyte co-expression. Methods: In this study, varying severities of murine models of critical illness were utilized to explore HVEM+BTLA+ co-expression in the thymic and splenic immune compartments, while circulating blood lymphocytes from critically ill patients were also assessed for HVEM+BTLA+ co-expression. Results: Higher severity murine models resulted in minimal change in HVEM+BTLA+ co-expression, while the lower severity model demonstrated increased HVEM+BTLA+ co-expression on thymic and splenic CD4+ lymphocytes and splenic B220+ lymphocytes at the 48-hour time point. Patients demonstrated increased co-expression of HVEM+BTLA+ on CD3+ lymphocytes compared to controls, as well as CD3+Ki67- lymphocytes. Both L-CLP 48hr mice and critically ill patients demonstrated significant increases in TNF-α. Discussion: While HVEM increased on leukocytes after critical illness in mice and patients, changes in co-expression did not relate to degree of injury severity of murine model. Rather, co-expression increases were seen at later time points in lower severity models, suggesting this mechanism evolves temporally. Increased co-expression on CD3+ lymphocytes in patients on non-proliferating cells, and associated TNF-α level increases, suggest post-critical illness co-expression does associate with developing immune suppression.

Application of single cell multiomics points to changes in chromatin accessibility near calcitonin receptor like receptor and a possible role for adrenomedullin in the post-shock lung.

Introduction: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a commonly occurring sequelae of traumatic injury resulting from indirect insults like hypovolemic shock and/or extrapulmonary sepsis. The high lethality rate associated with these pathologies outlines the importance of clarifying the "priming" effects seen in the post-shock lung microenvironment, which are understood to bring about a dysregulated or overt immune response when triggered by a secondary systemic infectious/septic challenge culminating in ALI. In this pilot project, we test the hypothesis that application of a single cell multiomics approach can elucidate novel phenotype specific pathways potentially contributing to shock-induced ALI/ARDS. Methods: Hypovolemic shock was induced in C57BL/6 (wild-type), PD-1, PD-L1, or VISTA gene deficient male mice, 8-12 weeks old. Wild-type sham surgeries function as negative controls. A total of 24-h post-shock rodents were sacrificed, their lungs harvested and sectioned, with pools prepared from 2 mice per background, and flash frozen on liquid nitrogen. N = 2 biological replicates (representing 4 mice total) were achieved for all treatment groups across genetic backgrounds. Samples were received by the Boas Center for Genomics and Human Genetics, where single cell multiomics libraries were prepared for RNA/ATAC sequencing. The analysis pipeline Cell Ranger ARC was implemented to attain feature linkage assessments across genes of interest. Results: Sham (pre-shock) results suggest high chromatin accessibility around calcitonin receptor like receptor (CALCRL) across cellular phenotypes with 17 and 18 feature links, exhibiting positive correlation with gene expression between biological replicates. Similarity between both sample chromatin profiles/linkage arcs is evident. Post-shock wild-type accessibility is starkly reduced across replicates where the number of feature links drops to 1 and 3, again presenting similar replicate profiles. Samples from shocked gene deficient backgrounds displayed high accessibility and similar profiles to the pre-shock lung microenvironment. Conclusion: High pre-shock availability of DNA segments and their positive correlation with CALCRL gene expression suggests an apparent regulatory capacity on transcription. Post-shock gene deficient chromatin profiles presented similar results to that of pre-shock wild-type samples, suggesting an influence on CALCRL accessibility. Key changes illustrated in the pre-ALI context of shock may allow for additional resolution of "priming" and "cellular pre-activation/pre-disposition" processes within the lung microenvironment.

Deep RNA sequencing of intensive care unit patients with COVID-19.

COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (> 100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies.

NLRP3 inflammasome activation in cigarette smoke priming for Pseudomonas aeruginosa-induced acute lung injury.

It is increasingly recognized that cigarette smoke (CS) exposure increases the incidence and severity of acute respiratory distress syndrome (ARDS) in critical ill humans and animals. However, the mechanism(s) is not well understood. This study aims to investigate mechanism underlying the priming effect of CS on Pseudomonas aeruginosa-triggered acute lung injury, by using pre-clinic animal models and genetically modified mice. We demonstrated that CS impaired P. aeruginosa-induced mitophagy flux, promoted p62 accumulation, and exacerbated P. aeruginosa-triggered mitochondrial damage and NLRP3 inflammasome activation in alveolar macrophages; an effect associated with increased acute lung injury and mortality. Pharmacological inhibition of caspase-1, a component of inflammasome, attenuated CS primed P. aeruginosa-triggered acute lung injury and improved animal survival. Global or myeloid-specific knockout of IL-1ß, a downstream component of inflammasome activation, also attenuated CS primed P. aeruginosa-triggered acute lung injury. Our results suggest that NLRP3 inflammasome activation is an important mechanism for CS primed P. aeruginosa-triggered acute lung injury. (total words: 155).

The Neonatal Innate Immune Response to Sepsis: Checkpoint Proteins as Novel Mediators of This Response and as Possible Therapeutic/Diagnostic Levers.

Sepsis, a dysfunctional immune response to infection leading to life-threatening organ injury, represents a significant global health issue. Neonatal sepsis is disproportionately prevalent and has a cost burden of 2-3 times that of adult patients. Despite this, no widely accepted definition for neonatal sepsis or recommendations for management exist and those created for pediatric patients are significantly limited in their applicability to this unique population. This is in part due to neonates' reliance on an innate immune response (which is developmentally more prominent in the neonate than the immature adaptive immune response) carried out by dysfunctional immune cells, including neutrophils, antigen-presenting cells such as macrophages/monocytes, dendritic cells, etc., natural killer cells, and innate lymphoid regulatory cell sub-sets like iNKT cells, γδ T-cells, etc. Immune checkpoint inhibitors are a family of proteins with primarily suppressive/inhibitory effects on immune and tumor cells and allow for the maintenance of self-tolerance. During sepsis, these proteins are often upregulated and are thought to contribute to the long-term immunosuppression seen in adult patients. Several drugs targeting checkpoint inhibitors, including PD-1 and PD-L1, have been developed and approved for the treatment of various cancers, but no such therapeutics have been approved for the management of sepsis. In this review, we will comparatively discuss the role of several checkpoint inhibitor proteins, including PD-1, PD-L1, VISTA, and HVEM, in the immune response to sepsis in both adults and neonates, as well as posit how they may uniquely propagate their actions through the neonatal innate immune response. We will also consider the possibility of leveraging these proteins in the clinical setting as potential therapeutics/diagnostics that might aid in mitigating neonatal septic morbidity/mortality.

Negative Immune Checkpoint Protein, VISTA, Regulates the CD4+ Treg Population During Sepsis Progression to Promote Acute Sepsis Recovery and Survival.

Sepsis is a systemic immune response to infection that is responsible for ~35% of in-hospital deaths and over 24 billion dollars in annual treatment costs. Strategic targeting of non-redundant negative immune checkpoint protein pathways can cater therapeutics to the individual septic patient and improve prognosis. B7-CD28 superfamily member V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an ideal candidate for strategic targeting in sepsis. We hypothesized that immune checkpoint regulator, VISTA, controls T-regulatory cells (Treg), in response to septic challenge, thus playing a protective role/reducing septic morbidity/mortality. Further, we investigated if changes in morbidity/mortality are due to a Treg-mediated effect during the acute response to septic challenge. To test this, we used the cecal ligation and puncture model as a proxy for polymicrobial sepsis and assessed the phenotype of CD4+ Tregs in VISTA-gene deficient (VISTA-/-) and wild-type mice. We also measured changes in survival, soluble indices of tissue injury, and circulating cytokines in the VISTA-/- and wild-type mice. We found that in wild-type mice, CD4+ Tregs exhibit a significant upregulation of VISTA which correlates with higher Treg abundance in the spleen and small intestine following septic insult. However, VISTA-/- mice have reduced Treg abundance in these compartments met with a higher expression of Foxp3, CTLA4, and CD25 compared to wild-type mice. VISTA-/- mice also have a significant survival deficit, higher levels of soluble indicators of liver injury (i.e., ALT, AST, bilirubin), and increased circulating proinflammatory cytokines (i.e., IL-6, IL-10, TNFα, IL-17F, IL-23, and MCP-1) following septic challenge. To elucidate the role of Tregs in VISTA-/- sepsis mortality, we adoptively transferred VISTA-expressing Tregs into VISTA-/- mice. This adoptive transfer rescued VISTA-/- survival to wild-type levels. Taken together, we propose a protective Treg-mediated role for VISTA by which inflammation-induced tissue injury is suppressed and improves survival in early-stage murine sepsis. Thus, enhancing VISTA expression or adoptively transferring VISTA+ Tregs in early-stage sepsis may provide a novel therapeutic approach to ameliorate inflammation-induced death.

Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations.

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.

Patho-Mechanisms for Hemorrhage/Sepsis-Induced Indirect Acute Respiratory Distress Syndrome: A Role for Lung TIE1 and Its Regulation by Neutrophils.

INTRODUCTION: Severe hemorrhage (Hem) has been shown to be causal for the development of extra-pulmonary/indirect acute respiratory distress syndrome (iARDS) and is associated with severe endothelial cell (EC) injury. EC growth factors, Angiopoietin (Ang)-1 and -2, maintain vascular homeostasis via tightly regulated competitive interaction with the tyrosine kinase receptor, Tie2, expressed on ECs. OBJECTIVE: Since it has been reported that the orphan receptor, Tie1, may be able to play a role in Ang:Tie2 signaling; we chose to examine Tie1's capacity to alter the lung Ang:Tie2 interaction in response to the sequential insults of shock/sepsis (cecal ligation and puncture [CLP]), culminating in iARDS. METHODS: Male mice were subjected to Hem alone or sequential Hem followed 24 hours later by CLP that induces iARDS. Changes in lung and/or plasma levels of Tie1, Tie2, Ang-1, Ang-2, various systemic cytokine/chemokines and indices of lung injury/inflammation were then determined. The role of Tie1 was established by intravenous administration of Tie1 specific or control siRNA at 1 h post-Hem. Alternatively, the contribution of neutrophils was assessed by pre-treating mice with anti-neutrophil antibody depletion 48 h prior to Hem. RESULTS: Lung tissue levels of Tie1 expression elevated over the first 6 to 24 h post-Hem alone. Subsequently, we found that treatment of Hem/CLP mice with Tie1-specific siRNA not only decreased Tie1 expression in lung tissue compared to control siRNA, but, suppressed the rise in lung inflammatory cytokines, lung MPO and the rise in lung protein leak. Finally, much as we have previously shown that neutrophil interaction with resident pulmonary vascular ECs contribute significantly to Ang-2 release and EC dysfunction, central to the development of iARDS. Here, we report that depletion of neutrophils also decreased lung tissue Tie1 expression and increased Tie2 activation in Hem/CLP mice. CONCLUSION: Together, these data imply that shock-induced increased expression of Tie1 can contribute to EC activation by inhibiting Ang:Tie2 interaction, culminating in EC dysfunction and the development of iARDS.

Emerging therapeutic targets for sepsis.

Introduction: Sepsis is characterized by a dysregulated host response to infection. Sepsis-associated morbidity/mortality demands concerted research efforts toward therapeutic interventions which are reliable, broadly effective, and etiologically based. More intensive and extensive investigations on alterations in cellular signaling pathways, gene targeting as a means of modifying the characteristic hyper and/or hypo-immune responses, prevention through optimization of the microbiome, and the molecular pathways underlying the septic immune response could improve outcomes.] Areas covered: The authors discuss key experimental mammalian models and clinical trials. They provide an evaluation of evolving therapeutics in sepsis and how they have built upon past and current treatments. Relevant literature was derived from a PubMed search spanning 1987-2020.Expert opinion: Given the complex nature of sepsis and the elicited immune response, it is not surprising that a single cure-all therapeutic intervention, which is capable of effectively and reliably improving patient outcomes has failed to emerge. Innovative approaches seek to address not only the disease process but modify underlying patient factors. A true improvement in sepsis-associated morbidity/mortality will require a combination of unique therapeutic modalities.

Survival and Pulmonary Injury After Neonatal Sepsis: PD1/PDL1's Contributions to Mouse and Human Immunopathology.

Morbidity and mortality associated with neonatal sepsis remains a healthcare crisis. PD1-/- neonatal mice endured experimental sepsis, in the form of cecal slurry (CS), and showed improved rates of survival compared to wildtype (WT) counterparts. End-organ injury, particularly of the lung, contributes to the devastation set forth by neonatal sepsis. PDL1-/- neonatal mice, in contrast to PD1-/- neonatal mice did not have a significant improvement in survival after CS. Because of this, we focused subsequent studies on the impact of PD1 gene deficiency on lung injury. Here, we observed that at 24 h post-CS (but not at 4 or 12 h) there was a marked increase in pulmonary edema (PE), neutrophil influx, myeloperoxidase (MPO) levels, and cytokine expression sham (Sh) WT mice. Regarding pulmonary endothelial cell (EC) adhesion molecule expression, we observed that Zona occludens-1 (ZO-1) within the cell shifted from a membranous location to a peri-nuclear location after CS in WT murine cultured ECs at 24hrs, but remained membranous among PD1-/- lungs. To expand the scope of this inquiry, we investigated human neonatal lung tissue. We observed that the lungs of human newborns exposed to intrauterine infection had significantly higher numbers of PD1+ cells compared to specimens who died from non-infectious causes. Together, these data suggest that PD1/PDL1, a pathway typically thought to govern adaptive immune processes in adult animals, can modulate the largely innate neonatal pulmonary immune response to experimental septic insult. The potential future significance of this area of study includes that PD1/PDL1 checkpoint proteins may be viable therapeutic targets in the septic neonate.

Deep RNA Sequencing of Intensive Care Unit Patients with COVID-19.

PURPOSE: COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. METHODS: Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (>100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. RESULTS: We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. CONCLUSIONS: Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies. TAKE HOME MESSAGE: Deep RNA sequencing provides a novel diagnostic tool for critically ill patients. Among ICU patients with COVID-19, RNA sequencings can identify gene expression, pathogens (including SARS-CoV-2), and can predict mortality. TWEET: Deep RNA sequencing is a novel technology that can assist in the care of critically ill COVID-19 patients & can be applied to other disease.

A Peptide-Based Checkpoint Immunomodulator Alleviates Immune Dysfunction in Murine Polymicrobial Sepsis.

ABSTRACT: Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.

Unmasking Unique Immune Altering Aspects of the Microbiome as a Tool to Correct Sepsis-Induced Immune Dysfunction.

Background: Sepsis-related mortality is driven by immune dysfunction. A bidirectional micro-organism-immune cell cross talks exists. Gut Bacteroides fragilis-T-cell crosstalk maintains innate immune cell/pathogen homeostasis. Commensal gut Clostridia spp. suppress inflammation and induce gut tolerance. Probiotics are administered to restore immune microbiome homeostasis. Individual microbial components have an immunomodulatory effect. However, probiotic therapies for sepsis-induced immune disruptions are rarely tailored to specific immune responses. Thus, we ask the question as to how components of the intestinal microbiome, often found in probiotic therapies, affect lymphocyte phenotypic profile? Methods: T-lymphocytes were cultured with either monomicrobial or polymicrobial combinations. Microbes used were Bacteroides fragilis, Clostridium perfringens, or Lactobacillus acidophilus. Cytokines, measured by enzyme-linked immunosorbent assay (ELISA)-included interleukin (IL)-6, IL-10, IL-22, and IL-33. Flow cytometry was used for T-cell phenotyping for program-death receptor-1 (PD-1) and B- and T-lymphocyte attenuator (BTLA). T-cell DNA was extracted to assess global epigenetic changes. For translation, IL-33 was measured from surgical intensive care unit (ICU) patients with sepsis with either monomicrobial or polymicrobial infection. Results: Lactobacillus consistently induced IL-22 and IL-33. Bacteroides fragilis induced IL-33 only under polymicrobial (pB) conditions. Within surgical ICU patients, IL-33 levels were higher in polymicrobial versus monomicrobial patients. PD-1+ expression was lowest with either monomicrobial Bacteroides fragilis or Bacteroides fragilis predominant polymicrobial context. Conversely Bacteroides fragilis exposure induced a distinct PD-1-high subpopulation. B- and T-lymphocyte attenuator-positive expression did not differ after individual microbes. Among polymicrobial conditions, Bacteroides fragilis predominant (pB) and Lactobacillus acidophilus predominant (pL) increased BTLA+ expression. DNA methylation was most increased in response to Clostridium perfringens in monomicrobial and in response to Bacteroides fragilis in polymicrobial conditions. Conclusion: Unique microbe/lymphocyte interactions occur. Bacteroides fragilis induced a T-cell phenotype consistent with potential long-term immune recovery. This work begins to discover how varying microbes may induce unique functional and phenotypic T-lymphocyte responses.

Peripheral vascular endothelial growth factor changes after transcranial magnetic stimulation in treatment-resistant depression.

OBJECTIVES: To determine if vascular endothelial growth factor (VEGF) changes with transcranial magnetic stimulation (TMS) in treatment-resistant major depressive disorder (MDD). METHODS: Serum from a naturalistic population of 15 patients with MDD was collected at baseline and after standard TMS treatment. VEGF concentration was determined via ELISA. Inventory of Depressive Symptomatology Self Report and Patient Health Questionnaire were used as a measure of depression symptom severity, clinical response and remission. Mann-Whitney U and Kendall's Tau Correlation were used for continuous variables. RESULTS: VEGF increased from pre- to post-TMS (+30.3%) in remitters whereas VEGF decreased in non-remitters (-9.87%) (P < 0.05). This same pattern was observed when comparing mean %change in VEGF between responders (+14.7%) and non-responders (-14.9%) (P = 0.054). Correlation was present between change in VEGF concentration (baseline to post) and change in Inventory of Depressive Symptomatology-Self Report at Tx30 (r = -0.371, P < 0.054), reflecting greater increases in VEGF linked to greater improvement in depressive symptoms following the standard 6-week course of TMS. CONCLUSION: Patients with a successful treatment with TMS had significantly greater increase in VEGF from baseline to after treatment compared to non-responders/non-remitters and a larger increase in VEGF was associated with greater improvement in depressive symptoms after TMS. This is the first report examining VEGF levels in depressed patients receiving TMS. This study provides correlative data supporting further investigation into VEGF's role as an important mediator in the processes underpinning TMS' antidepressant effects and as a potential biomarker of clinical outcomes.

SHP2 inhibitor PHPS1 ameliorates acute kidney injury by Erk1/2-STAT3 signaling in a combined murine hemorrhage followed by septic challenge model.

BACKGROUND: Hypovolemic shock and septic challenge are two major causes of acute kidney injury (AKI) in the clinic setting. Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major protein phosphatase tyrosine phosphatase (PTPs), which play a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. In this study, we explored whether SHP2 signaling contributed to development of AKI sequential hemorrhage (Hem) and cecal ligation and puncture (CLP) and whether inactivation of SHP2 through administration of its selective inhibitor, phenylhydrazonopyrazolone sulfonate 1 (PHPS1), attenuated this injury. METHODS: Male C57BL/6 mice were subjected to Hem (a "priming" insult) followed by CLP or sham-Hem plus sham-CLP (S/S) as controls. Samples of blood and kidney were harvested at 24 h post CLP. The expression of neutrophil gelatinase-associated lipocalin (NGAL), high mobility group box 1 (HMGB1), caspase3 as well as SHP2:phospho-SHP2, extracellular-regulated kinase (Erk1/2): phospho-Erk1/2, and signal transducer and activator of transcription 3 (STAT3):phospho-STAT3 protein in kidney tissues were detected by Western blotting. The levels of creatinine (Cre) and blood urea nitrogen (BUN) in serum were measured according to the manufacturer's instructions. Blood inflammatory cytokine/chemokine levels were detected by ELISA. RESULTS: We found that indices of kidney injury, including levels of BUN, Cre and NGAL as well as histopathologic changes, were significantly increased after Hem/CLP in comparison with that in the S/S group. Furthermore, Hem/CLP resulted in elevated serum levels of inflammatory cytokines/chemokines, and induced increased levels of HMGB1, SHP2:phospho-SHP2, Erk1/2:phospho-Erk1/2, and STAT3:phospho-STAT3 protein expression in the kidney. Treatment with PHPS1 markedly attenuated these Hem/CLP-induced changes. CONCLUSIONS: In conclusion, our data indicate that SHP2 inhibition attenuates AKI induced by our double-hit/sequential insult model of Hem/CLP and that this protective action may be attributable to its ability to mitigate activation of the Erk1/2 and STAT3 signaling pathway. We believe this is a potentially important finding with clinical implications warranting further investigation.

Novel Peptide-Based PD1 Immunomodulators Demonstrate Efficacy in Infectious Disease Vaccines and Therapeutics.

Many pathogens use the same immune evasion mechanisms as cancer cells. Patients with chronic infections have elevated levels of checkpoint receptors (e.g., programed cell death 1, PD1) on T cells. Monoclonal antibody (mAb)-based inhibitors to checkpoint receptors have also been shown to enhance T-cell responses in models of chronic infection. Therefore, inhibitors have the potential to act as a vaccine "adjuvant" by facilitating the expansion of vaccine antigen-specific T-cell repertoires. Here, we report the discovery and characterization of a peptide-based class of PD1 checkpoint inhibitors, which have a potent adaptive immunity adjuvant capability for vaccines against infectious diseases. Briefly, after identifying peptides that bind to the recombinant human PD1, we screened for in vitro efficacy in reporter assays and human peripheral blood mononuclear cells (PBMC) readouts. We first found the baseline in vivo performance of the peptides in a standard mouse oncology model that demonstrated equivalent efficacy compared to mAbs against the PD1 checkpoint. Subsequently, two strategies were used to demonstrate the utility of our peptides in infectious disease indications: (1) as a therapeutic in a bacteria-induced lethal sepsis model in which our peptides were found to increase survival with enhanced bacterial clearance and increased macrophage function; and (2) as an adjuvant in combination with a prophylactic malaria vaccine in which our peptides increased T-cell immunogenicity and the protective efficacy of the vaccine. Therefore, our peptides are promising as both a therapeutic agent and a vaccine adjuvant for infectious disease with a potentially safer and more cost-effective target product profile compared to mAbs. These findings are essential for deploying a new immunomodulatory regimen in infectious disease primary and clinical care settings.